Archives of Phytopathology and Plant Protection

February 2005; 38(1): 31 – 39

Antisporulant activity of leaf extracts of Indian plants

against Sclerospora graminicola causing downy mildew
disease of pearl millet

SALIGRAMA ADAVIGOWDA DEEPAK1, GYULA OROS2,

SYAGADADU GIRIYANNA SATHYANARAYANA1,
NANDINI PRATAP SHETTY1, HUNTRIKE SHEKAR SHETTY1, &
SHEENA SASHIKANTH3
1
Department of Studies in Applied Botany, University of Mysore, Manasagangotri, Mysore 570 006,
Karnataka, India, 2Department of Toxicology, Plant Protection Institute Hungarian Academy of Sciences,
1525 Budapest 114, Pf. 102, Hungary, and 3Department of Studies in Chemistry, University of Mysore,
Manasagangotri, Mysore 570 006, Karnataka, India

(Received 16 June 2004)

Abstract
Methanolic extracts of forty plant species commonly growing across India were collected and have been
screened for antisporulant activity against Sclerospora graminicola (Sacc.) Schroet., the causative agent of
pearl millet downy mildew. The collection represented 38 genera of 30 families. The methanolic extracts
of nine species did not show any effect, whereas the activity of the extracts of Clematis gouriana, Evolvulus
alsinoides, Mimusops elengi, Allium sativum and Piper nigrum were commensurable to that of the marketed
botanical fungicides. The extracts of 11 species (Agave americana, Artemisia pallens, Citrus sinensis,
Dalbergia latifolia, Helianthus annus, Murraya koenigii, Ocimum basilicum, Parthenium hysterophorus, Tagetes
erecta, Thuja occidentalis and Zingiber offinale) exhibited remarkable antisporulant effect even after 10-fold
dilution of the crude extracts while in the case of remaining 15 plants the crude extracts loosed activity after
10-fold dilution. The antisporulant activity of commercialised Azadirachta preparation (Nutri-Neem) was
more pronounced than that of Reynutria based one (Milsana) and Sabadilla (veratrin), however, these
botanical preparations held off the extracts of C. gouriana and E. alsinoides and synthetic fungicides.

Keywords: Plant extracts, antifungal activity, Clematis, Evolvulus, downy mildew, pearl millet

Introduction
Present control technologies of downy mildews discouple the pathogen’s life cycle mainly in
two points of ontogeny. The applied chemicals either destroy spores, preventing the infection
or inhibit the biotrophic thallus, anticipating the formation of new infective propagules. The
preventive control of Sclerospora graminicola, the causative agent of pearl millet downy mildew
(PMDM) meets difficulties. The short vegetation reduces the remediative capacity of its host
plant, and as a consequence of it, pesticides are accumulated in the grain [1] challenging to

Correspondence: H.S. Shetty, Department of Studies in Applied Botany, University of Mysore, Manasagangotri, Mysore 570 006,
Karnataka, India. E-mail: hss_uom@hotmail.com

ISSN 0323-5408 print/ISSN 1477-2906 online # 2005 Taylor & Francis Group Ltd
DOI: 10.1080/03235400400007558
32 S. A. Deepak et al.

application of synthetic chemicals after formation of tillers. The resistant to PMDM cultivars
were selected and some of them have been cultivated widely. The pathogen shows high
natural variation in aggressivity [2]; even progenies of the same oospore could be classified
into distinct pathotype groups [3]. Although the downy mildew tolerance of pearl millet can
be enhanced by diverse methods [4, 5] the possibilities of biocontrol measures [6] as well as
enhancement of plant resistance with chemical treatment [7] were presented, none of these
approaches resulted the economically acceptable level of control. Thus, the vulnerability of
the resistance to the disease has been a major cause of concern as even 10% disease incidence
cause economic loss threatening the net return [8]. The only tool for creditable control of this
endobiotrophic peronospora is the use of systemically acting acylanilide derivatives. However,
the calculability of management of pearl millet downy mildew has been threatened by
emergence of acquired tolerance to this group of chemicals in India [9].
Losses caused by PMDM urges development of alternative control agents. One approach to
discover newer antimildew compounds is to search for their presence in natural sources [10].
The defence strategies of plants against their pathogens are multitudinous, including the use
of antimicrobial chemicals that either can be de novo formed during the pathogenesis
(phytoalexins) or are constitutive components (phytoanticipins) of cells [11]. On the other
hand, pathogens have evolved mechanisms to evade these barriers that demand application of
various pesticides in crop production. Microbial species or strains that do not invade the plant
are usually more sensitive to the components of performed barriers than a viable pathogen of
this plant [12]. Consequently, phytoanticipins and precursors of phytoalexins can represent a
prospective tool for PMDM management [13].
In the present investigation, we have studied the antisporulant activity of methanolic
extracts of plants growing at almost all locations throughout pearl millet producing areas of
India. The inhibitory effect of extracts against S. graminicola was compared to that of
marketed fungicides and phytochemicals.

Materials and methods

Plant materials and reference compounds
This survey was carried out during vegetation period of the pearl millet in 2003 to identify the
easily available plants in pearl millet producing regions of India. A total of 40 species
representing 38 genera of 30 families were collected from districts Mysore (12.18 N – 76.42 E,
770 m above sea level (ASL)), Mandya (12.33 N – 76.54 E, 695 m ASL), Mercara (12.26 N –
75.47 E, 1145 m ASL) and Hassan (13.01 N – 76.10 E, 957 m ASL), and Gopalaswamy Hills,
Chamarajanagar district (11.56 N – 77.00 E, 780 – 1455 m ASL). The plants were identified
taxonomically and authenticated at the Herbarium, Department of Botany, Mysore (Table I).
Fungicide dimethomorph (Acrobat 50 wp, Shell, UK) was supplied by the manufacturer
whereas metalaxyl (Ridomil 25 wp, Ciba-Geigy, Swiss) was extracted from marketed product.
Veratrine mixture, digitonin and podophyllotoxin were purchased from Sigma-Aldrich
(USA). The commercial preparations Mikal 70 wp (Rhone Poulenc, France), Nutri Neem
1
(Nutri-Tech Solutions, Pty Ltd., Australia) and Milsana Bioprotectant Concentrate (KHH
BioSci, Raleigh, USA) were also used as a reference.

Test organisms
The downy mildew pathogen S. graminicola (pathotype 1) was isolated from naturally infested
pearl millet (Pennisetum glaucum (L.) R. Br., hybrid HB3) in Bogadi village of Mysore district
 Table I. Inhibitory effect of plant extracts on zoosporangium formation of Sclerospora graminicola.

Activity of extracted plant material

Methanolic extracts4

No. Species (Family)1 Origin2 Part used3 crude 1:9 1 : 99 Reported activity against fungi5

Strong
1 Clematis gouriana Roxb. (Ranunculaceae) MY T + + ++ + F36, F42, F43
2 Evolvulus alsinoides L. (Convolvulaceae)a,b HA T + + + +
3 Allium sativum L. (Liliaceae)a,b MY B + + ++ 7 F16, F23
4 Piper nigrum L. (Piperaceae)a,b MY S + + ++ 7 F16, F6, F9, F10, F20, F27, F30, F36, F38,
F42, F43
5 Mimusops elengi L. (Sapotaceae)b MY F ++ ++ 7
Remarkable
6 Agave americana L. (Agavaceae)a MY L + + + 7
7 Thuja occidentalis L. (Cupressaceae) MY L + + + 7
8 Artemisia pallens Wall. (Asteraceae) CH L + + + 7
9 Helianthus annuus L. (Asteraceae) MY S + + + 7
10 Parthenium hysterophorus L. (Asteraceae) MY L + + + 7
11 Tagetes erecta L. (Asteraceae) MY L + + + 7
12 Ocimum basilicum L. (Lamiaceae)a,b MY L + + + 7 F5, F12, F21, F25, F27, F41

Antimildew activity of plant extracts

13 Zingiber offinale Roscoe (Zingiberaceae) a,b MY R + + + 7 F6, F12, F19, F23, F38
14 Citrus sinensis (L.) Oesbeck. (Rutaceae) a,b MY P + + + 7 F31, F33
15 Murraya koenigii Spreng. (Rutaceae) MY L + + + 7 F37
16 Dalbergia latifolia Roxb. (Papilonaceae) ME L + + + 7
Weak
17 Euphorbia hirta L. (Euphorbiaceae)b MY L + + 7 7
18 Aloe vera L. (Liliaceae) a,b MY L + + 7 7
19 Artemisia parviflora Wight. (Asteraceae) CH T + + 7 7
20 Datura metel L. (Solanaceae)a MY L + + 7 7
21 Leucas aspera Spreng. (Lamiaceae) MY L + + 7 7 F8
22 Ocimum sanctum L. (Lamiaceae) a,b MY L + + 7 7 F8, F9, F12, F21, F36, F38
23 Eucalyptus globosus Labill. (Myrtaceae) a,b MY L + + 7 7 F2, F4, F10, F14, F19, F26, F28, F38
24 Azardirachta indica A. Juss (Meliaceae) a,b MY L + + 7 7 F3, F5, F6, F11, F13, F15, F17, F18, F19,
F21, F22, F24, F27, F32, F35, F40
25 Citrus limon L. (Rutaceae) a,b
MY L ++ 7 7 F9, F36

(continued).

33
 34
Table I. (continued).

Activity of extracted plant material

S. A. Deepak et al.
Methanolic extracts4

No. Species (Family)1 Origin2 Part used3 crude 1:9 1 : 99 Reported activity against fungi5

26 Calotropis gigantea (L.) W. T. Aiton (Asclepiadaceae)b MY L ++ 7 7 F8, F11, F21, F27

27 Santalum album L. (Santalaceae) a,b MY L ++ 7 7
28 Artemisia vulgaris L. (Asteraceae) CH L + 7 7
29 Chrysanthemum indicum L. (Asteraceae) MY L + 7 7
30 Strobilanthes heynenus Ness. (Acanthaceae) CH F+L + 7 7
31 Salix tetrasperma Roxb. (Salicaceae) MA L + 7 7 F1, F29
Inactive
32 Achyranthes aspera L. (Amaranthacea)b MY L 7 7 7 F12, F21
33 Bixa orellana L. (Bixaceae)a MY L 7 7 7 F41
34 Cassine glauca (Rottb.) Kuntze (Celastraceae) MY L 7 7 7
35 Cucurbita maxima Duchesne (Cucurbitaceae) MY L 7 7 7
36 Ixora coccinea L. (Rubiaceae) MY L 7 7 7 F39
37 Mimosa pudica L. (Mimosaceae)a MY L 7 7 7
38 Mirabalis jalapa L. (Nyctganiceae) MY L 7 7 7
39 Rosa indica L. (Rosaceae) a,b MY L 7 7 7 F7
40 Zizypus rugosa Lam. (Rhamnaceae) MY L 7 7 7 F36
1
The species marked with a and b are Ayurvedic plants and sold as common bazar medicines, respectively [15]. 2Specimens originate from Mysore (MY), Mandaya (MA),
Chamarajanagar (CH), Mercara (ME) and Hassan (HA) districts, respectively. 3Leaves (L), twigs (T), flowers (F), bulbs (B), rhizome (R), seeds (S) or peel (P) of the plant
were used for extraction. 4The plant material was extracted with equivalent to mass of methanol and the filtrate was concentrated 1:20. The resulted concentrate was applied
in the screening process directly or diluted with distilled water 1 : 9, 1 : 99 and 1 : 999 ratios, respectively. The antisporulant activity was evaluated by following scale; full
inhibition ( + + ), partial inhibition ( + ) and no inhibition ( 7 ). The thousand fold diluted extracts did not exhibited activity. 5The fungal species with reported sensitivity to
extracts of the given plants are as follows: F1-Alternaria alternata [16, 17], F2-A. solani [18], F3-A. tenuis [19], F4-A. triticina [18], F5-Aspergillus sp. [20, 21], F6-A. niger
[17, 22, 23], F7-Botrytis cinerea [24 – 26], F8-Ceratocystis paradoxa [27], F9-Cochliobolus miyabeanus [28], F10-Colletotrichum capsici [17], F11-C. lindemuthianum [18], F12-
C. musae [29 – 31], F13-Glomerella cingulata [26], F14-Didymella bryoniae [32], F15-Drechslera oryzae [19], F16-Fusarium spp. [33 – 35], F17-F. moniliforme [36], F18-F.
nivale [37], F19-F. oxysporum [18, 19, 31], F20-F. pallidoroseum [17], F21-F. proliferatum [29, 30], F22-F. solani [38], F23-F. udum [39], F24-Gaumanomyces graminis [37],
F25-Geotrichum candidum [40], F26-Helminthosporium oryzae [18], F27-Botryosphaeria rhodina [17, 29, 30], F28-Macrophomina phaseolina [18], F29-Melampsora rust [41],
F30-Penicillium citrinum [17], F31-P. digitatum [42, 43], F32-P. expansum [26], F33-P. italicum [42], F34-Phomopsis caricae-papayae [17], F35-Puccinia arachidis [44], F36-
Pyricularia oryzae [28, 45, 46], F37-Pythium species [47], F38-Rhizoctonia solani [18, 28, 48], F39-Saccharomyces cerevisiae [49], F40-Sphaerotheca fuliginea [37], F41-
Trichoderma [22], F42-Ustilago maydis [51], F43-U. nuda [51].
 Antimildew activity of plant extracts 35

(Karnataka state, India) during 1970 by Shetty. The strain was maintained on greenhouse
grown pearl millet plants before being used as inoculum for the experiments. The method was
described in detail by Safeulla [14].

Preparation of plant extracts

The collected plant material in fresh conditions was weighed in situ, then cooled and stored to
prevent losing water. Tissues were cut into pieces and triturated with methanol (50 g with
50 ml). The homogenate was allowed to settle for 1 h, then it was filtered through muslin
cloth and centrifuged at 10 000 rpm for 15 min. Then the supernatant was concentrated to
5 ml and stored at 0 – 48C in a closed container and used as a crude extract. Dilutions (1 : 9,
1 : 99 and 1 : 999) have been made with distilled water before application in both cases. Tween
20 was added (0.02%) as a wetting agent.

Determination of antiperonospora activity

Leaves with disease symptoms were collected from artficially infected plants grown in the field
and washed in distilled water, excess water was then removed. The leaves were cut into
&1 cm2 pieces which were subsequently immersed for 30 min into test solution (crude
extract and its 10-fold dilutions as well as 5, 0.5, 0.05, 0.005 and 0.0005% w/v of reference
substances). Treated pieces were then incubated 12 – 14 h in moist chambers (plastic trays
lined with wet filter paper) at 22 + 18C in the dark. To determine the intensity of sporulation,
a 0 – 2 scale was used where the proportion of leaf area covered by zoosporangia was graded as
follows: 0, no sporulation; 1, the sporulation was inhibited partially; and 2, the intensity of
sporulation was indistinguishable of that on untreated control pieces, respectively. All tests
were carried out in quadruplicates.

Results and discussion

Most of extracts inhibited the zoosporangium formation of S. graminicola. Scores of the
degrees of inhibition caused by these extracts are presented in Table I. Only extracts of nine
plants did not show any activity (species 32 – 40), whereas the extracts of C. gouriana and E.
alsinoides proved to be active on the level of reference compounds (Table II). Although the
extracts of the leaves of three other species (M. elengi, A. sativum and P. nigrum) also strongly
inhibited the sporulation after 10-fold dilution with water, they were less effective than the
formers. The crude extracts of 11 plants (species 6 – 16) totally inhibited the sporulation and
some activity remained after 10-fold dilution as well. The excellent activity of crude extracts
of remaining plants (species 17 – 27) was completely lost after 10-fold dilution, while crude
extracts of species 28 – 31 inhibited the sporulation incompletely. The marketed botanicals
(Nutri-Neem, Milsana and Sabadilla) and podophyllotoxin did not exhibit excellent activity
against PMDM contrary to glycosteroid digitonin (Table II).
Excepting few species (2, 5, 7, 9, 16, 19, 35 and 37) large number of data was reported on
the antimicrobial activities of tested plants, although the published data refer mainly on
responses of human associated microbes (Candida, Trychophyton, Esherichia coli and various
other bacteria) and minor part relates to phytopathogenic fungi (Table I). Moreover, S.
graminicola is taxonomically distant from majority of pathogens tested earlier. Concerning to
the antioomycete activity few data were available on the activity of plants examined
throughout of this study. The extracts from leaves or bulbs of various Allium species are
known to act against large numbers of pathogens [39], among them peronosporas which is in
36 S. A. Deepak et al.
Table II. Antisporulant activity of commercial fungicides and reference substances.

Treatment Concentration (%) of substances

Substances Form1 0.0005 0.005 0.05 0.5 5

Dimethomorph A – + + ++ ++
Metalaxyl A – + + ++ ++
Mikal B – – + + ++
Digitonin A – – + + ++
Podophyllotoxin A – – – + ++
Veratrin A – – – + +
Nutri-Neem B – – – + +
Milsana B – – – + ++

The antisporulant activity was evaluated by following scale; full inhibition (+ +), partial inhibition (+) and no
inhibition (7). 1A = 25% methanolous stock solution of active ingredients containing 1% of Tween 20 was used for
preparing dilution series. The methanol and Tween 20 did not exhibited any inhibitory effect alone when applied at
maximum doses (5 and 0.2%, respectively). B = Commercial preparations were used.

accordance with our data. Leaf extracts of M. koenigii could control Pythium damping off at
67% when applied via soil in tomato [47] as well as the efficacy of bark-debris of Eucalyptus
against Phytophtora sp. was demonstrated [49]. Broad spectrum of antifungal activity was
reported in the case of several test plants (9, 16, 21, 22, 26), however, in our tests only Z.
officinale and O. basilicum exhibited remarkable antisporulant effect while E. globosus, A. indica
and O. sanctum acted weakly. Analysing the activity scores of different plant extracts against S.
graminicola (Table I) in relation to their effects reported against various phytopathogenic
fungi, there was clear that the sensitivity spectrum of S. graminicola is entirely different.
Moreover, no relationship was revealed between taxonomic position of plants and
antiperonospora activity of their extracts.
The role of phytoalexins in defence mechanisms was intensively studied meanwhile to
constitutive compounds has been paid less attention [13]. The term ‘phytoanticipin’ was
proposed for description of the latter group in 1994 and includes all types of low molecular
weight antimicrobial metabolites other than phytoalexins that are supposed to play a role in
disease resistance of plants [11]. In our case tissues of healthy plants were collected. It can be
presumed that the extracted constitutive compounds of plants were responsible for
antisporulant effect, which was manifested in our experiments. Nevertheless, it is often
difficult to determine whether a molecule is constitutive or induced, as some of them may
normally be present in hardly detectable quantities, but dramatically increase in concentration
after infection. Moreover, the same compound may be preformed antifungal substance in one
species and a phytoalexin in another [13].
The results of the present work indicate that some of the tested plants are promising
candidates for PMDM management. The transmission of S. graminicola to new areas by wind
can be successfully stopped by inhibition of zoosporangium formation [52]. The species
providing active extracts (A. sativum, P. nigrum, C. gouriana, E. alsinoides and M. elengi) are
found in pearl millet growing areas and are well known plants as most of them are used for
various medical purposes [11]. Their utilization makes possible the efficient pest management
exploiting the local natural resource base. The use of extracts possessing with broad spectrum
of activity (species 1, 3 and 4) can inhibit whole pathogen complex [53] associated to pearl
millet as well. Moreover, the costs of treatment are low and the contamination with residual
amounts of pesticides can be avoided [54].
 Antimildew activity of plant extracts 37

The use of various extracts of plants in agricultural practices has advantages. The sources
can be find in millet producing areas and the extracts do not need further bioremediation.
Although utilisation of toxic solvent in preparations made on-farm is not advisable, the
technology of manufacturing is simple and the resulted detoxified preparation easy to transfer
to the farmers, and thus to promote sustained millet production [53]. The analysis of
chemical composition of methanolic extracts can direct to discovery of new antimildew
substances which might be promising lead compounds for development of new synthetic
molecules with antiperonospora activity.

Acknowledgement
This research was supported by the co-operation project ‘‘ Theoretical base of integrated pest
control methods’’ between Indian National Science Academy and Hungarian Academy of
Sciences (Grant No. 11). The authors thank the Project Coordinator (ICAR-AICPMIP
Project, Jodhpur, India) for laboratory and field facilities provided.

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